PROJECT SUMMARY Hepatocellular carcinoma (HCC) has an annual worldwide incidence of more than 600,000 cases and a mortality rate greater than 95%, and therefore new and more effective therapies are needed for HCC patients. Sphingolipids, particularly ceramides and sphingosine 1-phosphate (S1P), regulate critical aspects of tumor biology. The anti-apoptotic and pro-survival lipid S1P is generated by sphingosine kinases (SK1 and SK2), and there is strong evidence that SK2-generated S1P drives cancer cell proliferation. We have developed SK inhibitors, including ABC294640, that have in vivo anti-inflammatory and anticancer activities against a variety of cancer types, including HCC and prostate cancer. Mechanistically, our recently studies suggest that ABC294640 mediates tumor suppression at least in part by targeting c-Myc for proteasomal degradation. Because c-Myc is a critical driver of HCC, being overexpressed in most HCC tumors and correlating with enhanced tumor growth and poor prognosis, defining the molecular mechanism(s) for SK2-regulation of c-Myc activity is critical for optimizing the clinical activity of ABC294640 and other sphingolipid-targeted drugs. Importantly, we have successfully completed enrollment to the first-in-human Phase I clinical trial of ABC294640 at the Medical University of South Carolina in patients with advanced solid tumors, and the data demonstrate positive safety, pharmacokinetic and pharmacodynamic profiles in these patients. Of high importance, plasma levels of ABC294640 that decrease plasma S1P levels and that are predicted to have anticancer activity can be safely achieved in these patients. Based on our completed nonclinical and clinical studies, we hypothesize that inhibition of SK2/S1P by ABC294640 will mediate tumor suppression at least in part through inhibition of c-Myc expression. This novel hypothesis will be tested in the following Specific Aims: Specific Aim 1. Determine the mechanisms by which inhibition of SK2/S1P mediates tumor suppression via the regulation of c-Myc expression. In this Aim, we will test our mechanistic hypothesis that SK2-generated S1P protects c-Myc from proteasomal degradation, thereby allowing c-Myc activity, and increased tumor proliferation. As a corollary, we also hypothesize that inhibition of SK2/S1P signaling by ABC294640 results in proteasomal degradation of c-Myc, leading to tumor suppression. Specific Aim 2. To conduct a Phase II trial of ABC294640 in patients with advanced HCC. In this Aim, we will test our novel clinical hypothesis that ABC294640 will provide a treatment benefit to patients with advanced HCC, which will be associated with decreased c-Myc and S1P signaling in the tumor.